The synthesis of plasma membrane proteins by mouse L-cells will be investigated by the use of cell-free protein synthesizing systems and immunoprecipitation of the polypeptide products. Both the mechanism of insertion of different types of plasma membrane protein and the physiological control of membrane formation will be studied. Plasma membrane fractions will be isolated by several procedures with emphasis laid on methods which provide 'inside out' preparations. The protein composition of such preparations will be compared by polyacrylamide gel electrophoresis. Intact cells will be subjected to lactoperoxidase-catalysed iodination and galactose oxidase-NaBH4 treatment to label proteins exposed at the extracellular surface. The same labelling techniques together with protease digestion will be used on 'inside-out' plasma membrane preparations in order to identify transmembrane, extracellular-face and cytoplasmic-face proteins. Plasma membranes and specific proteins of known orientation will be isolated and used to prepare specific anti-sera. These anti-sera will be characterized and then used to precipitate plasma membrane proteins synthesized by intact cells, cell-free extracts and mRNA isolated from L-cells and translated in rabbit reticulocyte lysates. This procedure willthen be uued to determine the site of synthesis of different plasma membrane proteins by translating RNA extracted from free and membrane-bound polysomes. Total cellular RNA will also be translated in a reticulocyte lysate supplemented with stripped rough microsomes from rat liver. In this case the association of newly synthesized plasma membrane proteins with the microsomal membrane will be investigated by measuring trypsin sensitivity of proteins which can be immunoprecipitated by plasmalemma anti-sera. This approach will be extended to study the synthesis of intracellular membrane proteins. Anti-sera will be raised against several organelles isolated from mouse liver and used to immunoprecipitate peptides synthesized under the direction of L-cell RNA in a reticulocyte lysate. These procedures will also be used to study the level (transcriptional or translational) at which the synthesis of membrane proteins is controlled.